This invention relates to methods and compositions for altering the viability of cells, particularly nonnormal or nonhealthy cells, such as those comprising various cancers in animals and humans. More particularly, the invention relates to the preparation and use of the compositions of the present invention to rapidly kill or weaken cancerous cells within or on the surface of an animal or human body. The invention also relates to methods to minimize metastatic growth potential of cancer cells derived from primary tumors during surgical intervention. Primarily intratumoral, but also systemic or parenteral treatments as well as pre-operative, post-operative, or topical treatment of cancers are contemplated. The present invention illustrates surprising and rapid effectiveness and relative specificity against a broad class of cancer cells and cancerous tissues over healthy cells.
Despite vast advances in screening, lifestyle moderation, and therapeutic approaches, cancer continues to be one of the leading causes of death in the world. Drug toxicity, drug resistance, and the varying genetic backgrounds of different cancer sub-types have complicated the development of chemotherapeutics that are specifically cytotoxic to cancerous cells, yet minimally toxic to the patient.
Many currently available cancer treatments depend on the type, location, size and stage of the tumor, as well as numerous patient health-related issues. Typically, treatment may involve surgery, chemotherapy, and radiation, alone or in combination. Surgical removal of a cancerous tumor, for example breast tumors, is often desired in conjunction with chemotherapy and/or radiation, because the tumor may represent a burden to the patient""s body.
During surgery, tumor cells often escape into the blood stream, leading to secondary tumors. Also the surgeon or radiologist may not succeed in removing/destroying the whole cancer tissue, leading to local recurrences. Therefore, even after seemingly successful treatment of a primary tumor, local recurrences and metastasis, i.e., the spread of malignant tumors to secondary sites, often represent further threats to the patient. It would be desirous to have methods and compositions that minimize the risk of metastasis and local recurrences, while at the same time present minimal toxic burden to the patient.
There remains a need for compositions with enhanced specificity and rapid cytotoxicity for a broad range of nonnormal or nonhealthy cells over healthy cells. In particular there remains a need for compositions and methods to treat and to prevent primary cancers and metastases that could be employed both locally, at the site of a primary tumor, and/or systemically, but that would present minimal toxic burden to the patient. Because of the heterogeneity of cancers, there further remains a need for a method to produce and to screen a library of active compositions from which a particular composition capable of altering the viability of a wide spectrum of cancerous cell types and sub-types may be chosen.
It is an object of the present invention to provide methods and compositions that alter the viability of a wide spectrum of nonnormal or nonhealthy cells, particularly cancers, but preferably that minimally affect the viability of healthy human cells. It has been found that the compositions and methods of the present invention demonstrate a surprisingly rapid, relatively specific, cytotoxicity for a broad range of nonnormal cells, particularly cancers, including but not limited to breast (both estrogen receptor positive and negative), epidermal, melanomal, colorectal, bladder, cervical, neuroblastomal, prostate, ovarian, endometrial, and placental cancers.
In practice, the compositions of the present invention are formed from a set of components comprising one or more of the following: (A) a dithiocarbonyl, preferably dithiocarbamate, compound; (B) a divalent metal ion; (C) a modulator of cellular glutathione levels; and (D) an inhibitor of the phosphorylation of choline, which can significantly change the composition of biological membranes. The biologically effective compositions described herein induce a selective and rapid effect on the viability of preferably, nonnormal cells, by inducing apoptotic or necrotic cell death, or both, with the dominant pathway being apoptosis. Particularly preferred active compositions comprise all four components, although combinations of fewer components can be fully effective in certain tumors. Such compositions have been found to be useful against a broad spectrum of cancerous cell lines while exhibiting less toxicity to healthy cells. Such compositions are thereby useful to treat tumors in vivo that are typically heterogeneous. The multi-component embodiments can be thus effective against the entirety of such tumors, whereas conventional chemotherapies often have more limited specificity. Compositions comprising less than all four components are also able to alter the viability of one or more particular cell lines.
In one aspect of the invention, compositions of the present invention are prepared by incorporating one or more of the components A, B, C, and D into the composition. The dithiocarbonyl component A, preferably a dithiocarbamate compound, of the composition usually has the formula: (R1)m(R2)xe2x80x94Zxe2x80x94C(S)xe2x80x94Sxe2x80x94Y wherein m is 0 or 1, but other structures can be envisioned. For example, a dithiocarbamate moiety can be inserted into fatty acid chains, or between the phosphate group and the polar headgroup, or at the end of the polar headgroup in a phospholipid molecule.
Y is chosen from the group consisting of hydrogen, a pharmaceutically acceptable cation, a physiologically cleavable leaving group, a targeting moiety, or a pharmaceutically active drug, including a chemotherapeutic drug. In a preferred embodiment, Y represents a pharmaceutically acceptable cation, and may represent all or a portion of the component B listed above.
Z may be preferably chosen from either O or N, but if Z is O, then m=0. R1 and R2 may be independently chosen from the group consisting of hydrogen; or C1-C24 straight, branched, or cyclic alkyl, alkenyl, aryl, acyl, alkaryl, aralkyl, or alkoxy groups, optionally substituted with ester, ether, halogen, sulfate, hydroxy, or phosphate groups. R1 and R2 may be optionally connected via a bridge such as xe2x80x94(CH)nxe2x80x94, wherein n is 3-8, so that the resulting structure is heterocyclic, and may be optionally substituted on any of the carbon atoms of the ring. Representative substitutents include, for example, C1-C10 straight, branched, or cyclic alkyl, aryl, aryalkyl, or alkaryl groups, optionally substituted with hydroxy, halo, phosphate, sulfate, or sulfonate groups.
Preferred dithiocarbonyl compounds of the present invention include diethyldithiocarbamate (DEDC); tricyclo-[5.2.1.O2,6]-decyl(9[8]-xanthogenate (also known commonly as D609); tetraethylthiuram disulfide (also known commonly as Disulfuram, ((C2H5)2NCS2.]2)); and pyrrolidinedithiocarbamate (PDC). Other representative dithiocarbonyl compounds are set forth in U.S. Pat. No. 5,783,596, incorporated herein by reference.
The present compositions also may incorporate a metal ion component B. In particular, the dithiocarbonyl components of the present invention may be complexed with the metal cation component B. Suitable metal cations include Cu2+ and Zn2+. Most preferably, Zn2+ is employed.
The compositions of the present invention may also incorporate a component C to affect the intracellular redox state of nonnormal or nonhealthy cells. In particular, it has been surprisingly discovered that chemical and enzymatic modulators of cellular glutathione (GSH) levels enhance the efficacy of the compositions of the present invention. Those compounds that deplete cellular glutathione are preferred as component C, including ethacrynic acid (EA), L-buthionine-S,R-sulfoximine (BSO), diethylmaleate, 2-cyclohexene-1-one, and 1-chloro-2,4-dinitrobenzene (CDNB). Most preferably, EA is employed as component C, because in toxicological studies it appears to be relatively well tolerated by humans.
While not wishing to be bound by any theory, the component C of the present invention seems to affect the cellular concentration of reduced glutathione (GSH). GSH is perhaps the most important protective antioxidant against oxidants and electrophilic compounds. The GSH redox system is crucial in maintaining intracellular GSH/GSSG homeostasis. This system uses GSH as a substrate in detoxification of peroxides such as hydrogen peroxide and lipid peroxides, reactions which involve glutathione peroxidase. The reactions generate oxidized GSH (i.e., GSSG), which is reduced to GSH by glutathione reductase in a reaction requiring the hexose monophosphate-shunt pathway utilizing reduced nicotinamide adenine dinucleotide phosphate (NADPH). Glutathione also reduces nonenzymatically a number of substances, such as peroxides or free radicals.
Through a widely distributed enzyme, glutathione S-transferase (GST), GSH participates in the detoxification of many substances, i.e., organic halides, fatty acid peroxides, etc. In a GST-catalyzed reaction, GSH reacts with such compounds, followed by cleavage of GSH""s glutamyl and glycyl residues and then acetylation by acetyl-CoA to give a mercapturic acid. Ethacrynic acid (EA) and 1-Cl-2,4-dinitrobenzene are substrates for the GST-catalyzed conjugation reaction, resulting in the depletion of cellular GSH. Because the present compositions are likely to act via oxidative shock, depletion of cellular glutathione by these compositions inhibits the cellular defense system, and probably enhances the efficacy of the compositions.
It has been further found that inhibitors of the phosphorylation of choline, which is involved in the formation of the major membrane phospholipid phosphatidylcholine, in some cases surprisingly enhance the efficacy of the compositions of the present invention. Preferably, such an inhibitor comprises component D, and is incorporated into the compositions of the present invention. A preferred embodiment of component D is dimethylethanolamine (DMETN). A further advantage of using DMETN is that it also inhibits proliferation of several cancer cell lines (T-47D, 2R-75-1, A431, MDA-MB-231, AN3Ca, CaOV3) while generally increasing proliferation of normal cells. Therefore, should some cancer cells survive surgery, the presence of DMETN would be expected to inhibit their growth.
It is another object of the present invention to provide methods for preoperative and post-operative treatment of cancerous cells and metastases. Because the compositions of the present invention alter the viability of nonnormal cells with some selectivity, the compositions may be used to treat primary tumors as well as metastases. The compositions are designed mainly for intratumoral application, but they may be also used either systemically or parenterally (e.g., i.v., i.p., subcutaneously, topically, transdermally, etc.). The compositions also may be used to prevent metastatic spread of cancerous cells from a primary tumor by rapidly inducing apoptotic and/or necrotic cell death in the tumor prior to surgical intervention.
It is another object of the invention to provide methods for altering the viability of one or more nonnormal or nonhealthy cells by providing a composition according to the present invention and exposing the cells, preferably cancerous cells, to the composition. Exposure may be through systemic administration of the compositions, or parenterally through topical, transdermal, or, preferably, intratumoral administration.
It is a further object of the present invention to provide methods for generating libraries of active compositions from which a preferred active composition comprising one or more components A, B, C, and D may be chosen. The method is employed to generate a preferred composition that is specifically tailored to be maximally effective in altering the viability of a particular cancer, while preferably exhibiting minimal alteration to the viability of normal cells. Alternatively, the particular composition may be chosen to be more broadly effective against a range of cancers, while nonetheless preferably remaining minimally toxic to healthy cells. The method is comprised of steps wherein a set of components that includes at least two, preferably three, and most preferably all four of the components A, B, C, and D is provided for incorporation into a plurality of test compositions. The test compositions so generated may vary based on the components incorporated, their concentrations, and the like. The test compositions so generated are then examined to determine if any induces a desired biological effect against one or more nonhealthy or nonnormal cell types. Based on the results, the desired composition is selected from among said test compositions. The desired composition may be used, for example, to treat a heterogeneous cancerous tumor, or alternatively to treat a more homogeneous cancerous tumor.
Finally, it is yet another object of the present invention to provide methods to treat primary tumors as well as metastases by combining the compositions with other, already tested, chemotherapeutic agents including but not limited to: cyclophosphamide, doxorubicin, vinorelbine, cisplatin, paclitexel, topotecan, 5xe2x80x2-fluorouracyl, epirubicin, trimetrexate, etc. Such combination treatments may allow the use of these anticancer agents at lower concentrations, thereby decreasing their toxic side effects.
In addition, the present compositions may be used in combination with radiotherapy; again, such combinations may allow the use of lower dosage levels of the composition chemicals and lower levels of radiation, which would decrease any toxic side effects.